Detection and differentiation of mycobacterium tuberculosis complex bacteria by direct variant repeat oligotyping

ABSTRACT

A method and primary pair and kit for the in vitro amplification of nucleic acid using amplification primers in amplification reactions. There is used a pair of primers comprising oligonucleotide sequences complementary to a part of a Direct Repeat sequence of a microorganism belonging to the M tuberculosis complex of microorganisms, whereby hybridization to a Direct Repeat occurs and subsequently elongation of the hybridized primer takes place. The primers are such that elongation in the amplification reaction occurs for one primer in a 5&#39; direction and for the other primer in a 3&#39; direction.

INTRODUCTION

Tuberculosis is an infectious disease that yearly kills more people thanany other single infectious disease. The WHO estimates that yearly about10 million people contract tuberculosis and that 3 million people diefrom this disease (37). After a long period of slow decrease inincidence, tuberculosis is on the increase again in most Westerncountries. Furthermore, the emergence of multidrug-resistant M.tuberculosis strains and the association of tuberculosis and HumanImmunodeficiency Virus infected individuals are worsening the situationdramatically (1, 2, 4, 6, 7, 10, 37).

One of the key factors in the control of tuberculosis is the rapiddiagnosis of the disease and the identification of the sources ofinfection. M. tuberculosis strain typing has already proved to beextremely useful in outbreak investigations (6, 14, 33) and is beingapplied to a variety of epidemiologic questions in numerouslaboratories. Traditionally, laboratory diagnosis is done by microscopy,culturing of the micro-organism, skin testing and X-ray imaging.Unfortunately, these methods are often not sensitive, not specific andare very time-consuming, due to the slow growth rate of M. tuberculosis.Therefore, new techniques like in vitro amplification of M. tuberculosisDNA have been developed to rapidly detect the micro-organism in clinicalspecimens (14). The ability to differentiate isolates of M. tuberculosisby DNA techniques has revolutionarized the potential to identify thesources of infection and to establish main routes of transmission andrisk factors for acquiring tuberculosis by infection (1, 3-10, 14, 16,19-22, 25, 26, 29-36). The use of an effective universal typing systemwill allow strains from different geographic areas to be compared andthe movement of individual strains to be tracked. Such data may provideimportant insights and identify strains with particular problems such ashigh infectivity, high virulency and/or multidrug resistance. Analysisof large numbers of isolates may provide answers to long-standingquestions regarding the efficacy of BCG vaccination and the frequency ofreactivation versus reinfection.

Because M. tuberculosis complex bacteria constitute a geneticallyremarkably homogenous group of bacteria, repetitive DNA elements andtransposable elements, that are associated with genetic rearrangementsof chromosomal DNA, have been exploited for strain differentiation of M.tuberculosis. Two of these are insertion sequences and the remainder areshort repetitive DNA sequences with no known function or phenotype.

The most widely used element for strain differentiation is IS6110, a1355 bp insertion sequence, which was initially identified in M.tuberculosis (19, 30) and subsequently found to be distributed throughall M. tuberculosis complex bacteria, including Mycobacterium bovis,Mycobacterium africanum, M. microti and bovis BCG (11, 14, 15). Otherelements to potentially differentiate M. tuberculosis include the MajorPolymorphic Tandem Repeat (MPTR), the Polymorphic GC rich repetitivesequence (PGRS) and the Direct Repeat (DR) sequence (15, 16, 26).

Most methods described for strain differentiation of M. tuberculosisdepend on the so-called Restriction Fragment Length Polymorphism (RFLP)observed by the technique of Southern blotting. This technique requiresthe purification of chromosomal DNA from cultured M. tuberculosisbacteria. In addition this method is not suited for detecting a largenumber of strains, i.e. strains containing only one IS6110 copy or noIS6110 copy (35) when IS6110 fingerprinting is carried out as thepresence of multiple IS6110 units is required for RFLP. Virtually all M.bovis BCG strains as well as a number of strains from India (35) containonly a single IS copy. Most strains could however be differentiated byfingerprinting with the 36-bp direct repeat or the polymorphic GC-richrepetitive DNA-element. Less discriminative power was obtained with themajor polymorphic tandem repeat and the insertion element IS1081.Furthermore the known techniques of fingerprinting are demanding interms of costs, the technical skills, and the time needed to performthem successfully (32). Therefore, this way of "DNA fingerprinting"cannot be performed on a routine basis in most laboratories.

Although Polymerase Chain Reaction (PCR)-based methods have beendeveloped to increase the speed of M. tuberculosis fingerprinting, themethods still need the purification of DNA from cultured cells and/orare technically demanding (12, 13, 23, 24, 27).

Groenen et al. (12) describe a method of strain differentiation based onthe nature of the DNA polymorphism in the DR cluster enabling typing ofindividual M. tuberculosis strains in a single PCR. The described methodwas based on the genetic variation in the DR region and the PCR methodused the primers SP24-R (derived from spacer region 24) and IS-L(derived from the IS copy) on the basis of the previously establishedpartial sequence of the DR region in M. bovis BCG P3 (15). The sizes ofthe amplified products range from approximately 300 to 550 bp. Usingthis method it was illustrated that M. bovis strain 42 differs from M.bovis PCG P3 in the absence of a discrete DVR, namely DVR 26. M.tuberculosis strains H37Ra and H37Rv differed from the P3 strain in theabsence of two discrete contiguous DVR's. DVR 25 and DVR 26. The threeremaining M. tuberculosis isolates 1430 en 31 differed from M. bovis BCGp3 in the absence of a 262 bp stretch of DNA located directly left fromthe inverted repeat of IS6110 comprising DVR 27 to DVR 29, 15 bp of theunique spacer in DVR 26 and 18 bp of the DR in DVR 30. In the DVR-PCRmethod the method of Jeffreys et al. 1991 (17) was modified. In theJeffreys et al. method designated as MVR-PCR or digital typing advantageis taken of a frequently occuring polymorphism of a single base pair inthe 29 bp minisatelite repeat MS32. In the MVR-PCR two primer pairs areused each of which allows the amplification of MS32 multimers whicheither have an adenine (A) or a guanine (G) at the 5' terminus.Separation by electrophoresis of the amplified products allows thereading of the sequence of the MVR's containing either an A or G at the5' end respectively. In the DVR-PCR method the polymorphism in the DRregion mainly comprises the presence or absence of DVR's which like theMS32 minisateLlite are composed of a non-variant part (DR) and a variantpart (the spacer sequence). The MVR-PCR method was modified to permitthe selective amplification of multimers of DVR's containing either anA, C, G or T at the 5' end of the spacer at the junction with the DR.For this purpose four primer combinations were prepared to drive thefour spacer specific PCR's. Each combination contained the reverseprimer IS-L and either one of the four primers based on the DR sequence.These four primers designated DRA-R, DRC-R, DRG-R and DRT-R,respectively contained a sequence of 19 residues derived from theconserved DR sequence plus either one of the four bases at the 3'terminus. The principle of the method is shown in FIG. 3 of reference12. Each of the four DVR specific primers results in a ladder of DVRmultimers increasing inside from bottom to top. This results in aso-called first spacer residue sequence or FSR sequence.

Despite the excellent differentiation by DVR-PCR of the four strainsanalyzed in (12) the method has a number of disadvantages. The DNAsequence technique in the adapted version as described by Jeffreys etal. is technically extremely difficult. Often the ladder cannot be readvery well and as small fragments amplify better than the largerfragments the ladder is often incomplete. Furthermore, the test cannotbe carried out in a routine manner in a simple laboratory such as, forexample, a hospital laboratory. Therefore, in practical hospital testssuch a method cannot be used. Furthermore, apart from the sequencingproblems a Southern blot is also required which involves a large amountof work. The practical problems associated with Southern blothybridisation technique include the relatively complex nature of themethod which requires multiple steps over a number of days and a lengthydelay from isolation of the organism to the DNA typing result, largelybecause of cultivation of the organism in liquid media for DNAextraction. A rapid and simple means of strain typing, based on PCRamplification would circumvent delays in obtaining a typing result andprovide a relatively simply assay that could be performed in manylaboratories. To have sufficient genomic DNA for fingerprinting theisolate has to be subcultured for 2 to 4 additional weeks afteridentification. In the setting of an outbreak especially one ofmultidrug-resistant tuberculosis (MDRTB) rapid identification of stainsmay enhance control efforts by detection and interruption oftransmission chains.

In ref. 27 Ross and Dwyer disclose using the ends of the insertionsequence IS6110 as oligonucleotide primers in an attempt to amplify DNAbetween clusters of this element on the genome. This test is based onthe assumption that the insertion sequence IS6110 is present in 1 to 19copies on the genome and is located in different sites for variousstrains. They illustrate that the PCR amplification method disclosedproduced no clear product for the two strains without IS6110 therebyillustrating the disadvantage of this method. Furthermore, Ross andDwyer illustrate in their article that the PCR amplification using theends of the insertion sequence IS6110 resulted in bands of variousintensities, thereby illustrating the lack of reliability of the resultsof this test.

In ref. 13 a variant on the RFLP typing using the polymerase chainreaction with a primer specific for IS66110 is described wherein asecond primer complementary to a linker ligated to the restrictedgenomic DNA is used. In one strand the linker contains uracil in placeof thymidine and specific amplification is obtained by elimination ofthe strand with uracil-N-glycosylase. The same disadvantages for thismethod can be mentioned as for the previous PCR-DVR method.

In ref. 25 Palittapongarnpim et al. describe the use of PCR usingarbitrary primers. In this article it is illustrated that the PCRbanding patterns of the strains H37Rv and H37Ra are identical. Theyillustrated that arbitrarily primed PCR can distinguish strains of M.tuberculosis however the inability of APPCR to distinguish between theH37Rv and H37Ra strains demonstrates a limitation of the APPCR forcloser related strains.

This invention describes a method to differentiate microorganismsbelonging to the M. tuberculosis complex by a robust method, which israpid and simple. The method can be performed in a laboratory withoutsophisticated equipment and it can be carried out by technicians, who donot have to be trained in sophisticated molecular biological techniques.In addition, the method is suitable to simultaneously detect M.tuberculosis directly in clinical specimens and to type themicro-organisms, without the requirement to culture the slow-growingbacteria of the M. tuberculosis complex. Finally, in contrast to othermethods of M. tuberculosis strain differentiation used sofar, theinvention allows an easy and robust classification of different DNAtypes, without the need of sophisticated image processing software.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the DNA polymorphism found at a uniquechromosomal locus, the "Direct Repeat" (DR) region, which is uniquelypresent in M. tuberculosis complex bacteria. This locus was discoveredby Hermans et al. (15) in M. bovis BCG, the strain used worldwide tovaccinate against tuberculosis. The DR region in M. bovis BCG consistsof directly repeated sequences of 36 base pairs, which are interspersedby non-repetitive DNA spacers, each 35 to 41 base pairs in length (15).The number of copies of the DR sequence in M. bovis BCG was determinedto be 49. In other strains of the M. tuberculosis complex the number ofDR elements was found to vary (15). The vast majority of the M.tuberculosis strains contain one or more IS6110 elements in the DRcontaining region of the genome.

The recent study (12) described above showed that the genetic diversityin the DR region is generated by differences in the DR copy number,suggesting that homologeous recombination between DR sequences may be amajor driving force for the DR-associated DNA polymorphism (12). Thehigh degree of DNA polymorphism within a relatively small part of thechromosome makes this region well-suited for a PCR-based fingerprintingtechnique.

The invention described below is based on a unique method of in vitroamplification of DNA sequences within the DR region and thehybridisation of the amplified DNA with multiple, short syntheticoligomeric DNA sequences based on the sequences of the unique spacerDNA's within the DR region (FIG. 2). This differs from previous PCRmethods in the use of a set of primers with both primers having multiplepriming sites as opposed to having one of the primers bind to a fixedpriming site such as to a part of IS6110. Because M. tuberculosiscomplex strains differ in the presence of these spacer sequences,strains can be differentiated by the different hybridisation patternswith a set of various spacer DNA sequences.

Determination of the DNA sequence of the complete DR region in M.tuberculosis.

FIG. 1 depicts the structure of the DR region of M. bovis BCG asdetermined previously by Hermans et al. and Groenen et al. (12, 15). Forthe sake of convenience we will designate a DR plus its 3' adjacentspacer sequence as a "Direct Variant Repeat" (DVR). Thus, the DR regionis composed of a discrete number of DVR's, each consisting of a constantpart (DR) and a variable part (the spacer).

The sequenced part of the DR region in M. bovis BCG is printed in blackand it comprises 21 DVR's, 7 located 5' of the IS6110 element and 14DVR's 3' of the IS6110 element (including the one in which the IS6110element is located). The non-sequenced part is depicted in gray.

To determine the sequence of more spacers, we sequenced in thisinvention the sequence of the chromosomal region comprising the whole DRregion of M. tuberculosis H37Rv and also sequences flanking the DRregion. For this purpose we used cosmid T211 (obtained from Dr. Cole,Institut Pasteur, Paris), carrying the whole DR region. This cosmidcontains an insert of about 35 kb from M. tuberculosis strain H37Rv. Aphysical map was constructed and the stretch containing the DR regionwas localized by Southern blotting. Subclones were prepared and the DNAflanking the IS6110 element residing in the DR cluster was sequenced.The sequence is given in FIG. 3 (SEQ ID NO: 1).

As shown schematically in FIG. 1, the number of DR's in strain H37Rvamounts to 41. As previously found in M. bovis BCG, again each DR wasfound to be interspersed by a unique spacer sequence, varying in sizefrom 29 to 41 base pairs. the sequence of 13 DVR's of H37Rv areidentical to 13 DVR's in the previously sequenced homologous chromosomalregion of M. bovis BCG (15). The DVR's of H37Hv are numbered from 1 to41. the numbering beginning from 5' terminal DVR. The identical DVR'sare spacers 12 to 32.

The subject invention is directed at a method of in vitro amplificationof nucleic acid using amplification primers in a manner known per se inamplification reactions such as PCR, LCR or NASBA, wherein a pair ofprimers is used comprising oligonucleotide sequences sufficientlycomplementary to a part of the Direct Repeat sequence of a microorganismbelonging to the M. tuberculosis complex of microorganisms forhybridisation to a Direct Repeat to occur and subsequently elongation ofthe hybridized primer to take place, said primer being such thatelongation in the amplification reaction occurs for one primer in the 5'direction and for the other primer in the 3' direction. Due to themultiple presence of Direct Repeats in the microorganisms to be detectedthe use of such primers implies that all the spacer regions will beamplified in an efficient manner. In particular it is not necessary forextremely long sequences to be produced in order to obtain amplificationof spacers located at a distance from the primer. With the instantselection of the primer pairs a heterogenous product is obtainedcomprising a lot of smaller fragments all comprising spacer regionnucleic acid. Subsequently the detection of the amplified product canoccur simply by using an oligonucleotide probe directed at one or moreof the spacer regions one wishes to detect. In order to avoid hindrancein the amplification reactions the primers can have oligonucleotidesequences complementary to non-overlapping parts of the Direct Repeatsequence so that when both primers hybridize to the same Direct Repeatand undergo elongation they will not be hindered by each other. Inparticular to avoid any hindrance during elongation reactions when oneprimer DRa is capable of elongation in the 5' direction and the otherprimer DRb is capable of elongation in the 3' direction the DRa isselected such that it is complementary to a sequence of the DirectRepeat located to the 5' side of the sequence of the Direct Repeat towhich DRb is complementary. In a method according to the invention theprimer used must have an oligonucleotide sequence capable of hybridisingto the consensus sequence of the Direct Repeat in a manner sufficientfor amplification to occur under the circumstances of the particularamplification reaction. A person skilled in the art of amplificationreactions will have no difficulty in determining which length and whichdegree of homology is required for good amplification reactions tooccur. The consensus sequence of the Direct Repeat of microorganismsbelonging to the M. tuberculosis complex is given in FIG. 1. (SEQ ID NO:2)

The invention is also directed at a method of detection of amicroorganism belonging to the M. tuberculosis complex ofmicroorganisms, comprising

1) amplifying nucleic acid from a sample with the method described abovein any of the embodiments disclosed, followed by

2) carrying out a hybridisation test in a manner known per se, whereinthe amplification product is hybridised to an oligonucleotide probe or aplurality of different oligonucleotide probes, each oligonucleotideprobe being sufficiently homologous to a part of a spacer of the DirectRegion of a microorganism belonging to the M. tuberculosis complex forhybridisation to occur to amplified product if such spacer nucleic acidwas present in the sample prior to amplification, said hybridisationstep optionally being carried out without prior electrophoresis orseparation of the amplified product and

3) detecting any hybridised products in a manner known per se.

The detection method according to the invention can be carried out in alarge number of embodiments which will depend on the objective of thedetection method. For example, the method can be carried out by using anumber of oligonucleotide probes in the hybridisation test, said numbercomprising at least a number of oligonucleotide probes specific for thetotal spectrum of microorganisms it is desired to detect. For example,one can use oligonucleotide probes of spacer regions known to be presentin all microorganisms belonging to the M. tuberculosis complex. Use ofone such oligonucleotide probe will suffice to detect whether infectionwith a M. tuberculosis microorganism has occurred. It is also possibleto use a combination of oligonucleotide probes specific for certaintypes of M. tuberculosis complex microorganisms. For example, 13 spacerregions of the strain M. tuberculosis H37Rv have been found to be sharedwith M. bovis BCG. However, a large number of spacers from both types ofmicroorganisms differ. It is therefore possible to select specificcombinations of oligonucleotide probes in order to differentiate betweenthe various strains. As the majority of tuberculosis infections are dueto infections with microorganisms from the groups M. tuberculosis, M.bovis and M. africanum a method for detection of a microorganismaccording to the invention will preferably be directed at detection ofthe presence of such microorganisms. The spacer sequences of M.tuberculosis H37Rv and the spacer sequence of the M. bovis BCG have beendetermined. M. tuberculosis H37Rv comprises 41 spacer sequences and thesequences are given elsewhere in the text as sequence id. nos. 3 to 43M. bovis BCG spacer sequences are described in (15) by Hermans et al. InFIG. 2 of the cited reference Direct Repeats 24-43 are disclosed for M.bovis BCG strain 44 containing IS987. The intermediate spacer regionsequences are also provided in this figure. The sequence data of thecited reference have appeared in the EMBL Genbank and DDBJ NucleotideSequence Databases under the accession number X57835. The spacer regionsthat have been found to be common for M. tuberculosis H37Rv and M. bovisBCG are the spacers 20 to 32 of M. tuberculosis H37Rv.

A method according to the invention as disclosed in any of theembodiments above can be carried out using an oligonucleotide probebeing a sequence complementary to any of the spacer sequences of M.tuberculosis H37Rv or any of the spacer sequences of M. bovis BCG or asequence complementary to fragments or derivatives of said spacersequences, said oligonucleotide probe being capable of hybridising tosuch a spacer sequence and comprising at least seven consecutivenucleotides homologous to such a spacer sequence and/or exhibiting atleast 60% homology, preferably exhibiting at least 80% homology withsuch a spacer sequence and being at least 7 nucleotides long. Inparticular if one wishes to detect the presence of either M.tuberculosis H37Rv or M. bovis BCG any of the common spacer sequencescan be used for providing a suitable oligonucleotide probe for a methodaccording to the invention.

The invention is also directed at a method for differentiating the typeof microorganism belonging to the M. tuberculosis complex in a sample,in particular at a method wherein the sample is a clinical specimen. Themethod can be carried out on a sample without the cells from the samplehaving to be cultured for analysis to be carried out. Such a methodcomprises carrying out the detection method according to the inventionas disclosed above, followed by comparison of the hybridisation patternobtained with a reference. The reference can be the hybridisationpattern obtained with one or more strains of microorganism belonging tothe M. tuberculosis complex of microorganisms in an analogous manner tothat of the sample. Another possibility is to examine the result whereinthe reference is a source providing a list of spacer sequences andsources thereof, such as a data bank. Through predetermined analysis ofsuch a data bank and specific selection of oligonucleotide probes adifferentiating test can be provided specifically suited to themicroorganism strain or strains one wishes to differentiate between. Inthe example illustrating the invention 77 clinical samples were analysedusing a large number of oligonucleotide probes and an illustration ofthe types of hybridisation patterns that can be expected with a methodaccording to the invention is given. Due to the specific nature of thespacer regions and the specific combination of spacer regions in variousstrains these spacer regions are especially suited for differentiatingtests. This is why such spacer sequences from the template for designingoligonucleotide probes, suitable in a detection method ordifferentiating method according to the invention. The invention istherefore also directed at oligonucleotide probes of at least 7nucleotides, preferably more than 12 nucleotides, in particularcomprising between 12 to 40 nucleotides, said probe being sufficientlyhomologous to any of the following spacer sequences; spacer sequences1-23 of M. bovis BCG, spacer sequences 44-49 of M. bovis BCG and spacerregions 1-43 of M. tuberculosis H37Rv with the exception of the M.tuberculosis H37Rv sequences common to M. bovis BCG. i.e. with theexception of any spacer regions corresponding to numbers 20-32 of M.bovis BCG. In particular Sequence id. No's 3-21 and 35-43 fall withinthe scope of the invention. The invention is also directed at fragmentsor derivatives of such spacer sequences capable of hybridising to such aspacer sequence, said oligonucleotide probe being at least 7oligonucleotides long, preferably more than 12 nucleotides, inparticular comprising between 12 to 40 nucleotides and comprising atleast 7 consecutive nucleotides homologous to such a spacer sequenceand/or exhibiting at least 60% homology, preferably exhibiting at least80% homology, most preferably exhibiting more than 90% homology with thecorresponding part of the spacer sequence.

The invention is also directed at a carrier comprising oligonucleotideprobes comprising at least one oligonucleotide probe wherein theoligonucleotide probe is specific for a spacer region of a microorganismof the group belonging to M. tuberculosis complex. In particular at acarrier comprising an oligonucleotide probe according to the inventionas disclosed above.

The invention is also directed at a pair of primers wherein both primerscomprise oligonucleotide sequences sufficiently complementary to a partof the Direct Repeat sequence of a microorganism belonging to the M.tuberculosis complex of microorganisms for hybridisation to occur andsubsequently elongation of the hybridised primer to take place, saidprimers being such that elongation in the amplification reaction occursfor one primer in the 5' direction and for the other primer in the 3'direction and wherein sufficiently complementary means saidoligonucleotide sequence comprises at least seven consecutive nucleotidehomologous to such a Direct Repeat sequence, in particular the consensussequence of a Direct Repeat (sequence id. no. 2) and/or exhibits atleast 60% homology, preferably exhibits at least 80% homology, mostpreferably exhibits more than 90% homology with the corresponding partof the direct repeat sequence and is at least 7 oligonucleotides long.In particular the primer pair DRa and DRb described in the example are aprimer pair suitable for carrying out the invention. A primer pair asdisclosed comprising one primer DRa capable of elongation in the 5'direction and the other primer DRb capable of elongation in the 3'direction with DRa being complementary to a sequence of the DirectRepeat located to the 5" side of the sequence of the Direct Repeat towhich DRb is complementary, the Direct Repeat being present in theDirect Region of a microorganism belonging to the group of M.tuberculosis complex falls within the scope of the invention.

A kit for carrying out a method for in vitro amplification of nucleicacid using amplification primers in a manner known per se inamplification reactions such as PCR, LCR or NASBA wherein a pair ofprimers is used comprising oligonucleotide sequences sufficientlycomplementary to a part of the Direct Repeat sequence of a microorganismbelonging to the M. tuberculosis complex of microorganisms forhybridisation to a Direct Repeat to occur and subsequently elongation ofthe hybridised primer to take place, said primers being such thatelongation in the amplification reaction occurs for one primer in the 5'direction and for the other primer in the 3' direction is an embodimentof the invention. Such a kit must comprise a suitable primer pair asdisclosed according to the invention. The kit of the invention can alsobe suitable for carrying out a method of detection of a microorganismbelonging to the M. tuberculosis complex of microorganisms as described.Such a kit comprises a primer pair as disclosed for the amplificationmethod and an oligonucleotide sequence as disclosed being sufficientlyhomologous to a spacer sequence of a Direct Region of a microorganismbelonging to the M. tuberculosis complex or a carrier comprising such anoligonucleotide sequence in any of the embodiment disclosed in thedescription for detection and differentiation.

EXAMPLE

In vitro amplification of the DR-containing region in clinical isolatesof M. tuberculosis.

The chromosomal DR region of 74 different clinical isolates of M.tuberculosis was amplified by the polymerase chain reaction (PCR), usingthe primer pair DRa (with Sequence id. no. 50) and DRb (with Sequenceid. no. 51). As illustrated in FIG. 4, a reaction product was obtainedfrom all strains investigated and the amplified DNA was heterogenous insize. This heterogeneity is to be expected, because the primers DRa andDRb can initiate the PCR at any of the DVR's in the DR region. Thereforeeach of the DVR's is expected to be present in the amplified PCRproduct. A good amplification is obtained in particular for the spacerregions at the termini of the direct region in contrast to the known PCRamplification reaction using nucleic acid of the IS fragment as primer(15 and 34).

Hybridisation of the amplified DR region to individual spacer sequencesof H37Rv.

The PCR products of the 74 above-mentioned strains were hybridized to 47spacer sequences, which were covalently bound to Biodyne C paper asdescribed (18). Because the PCR products contained a biotin label, whichwas incorporated during the PCR, hybridizing DNA could be visualized bybinding of a strepavidin-containing peroxidase conjugate and an enzymeassay. The result is shown in FIG. 5. DVR-amplified DNA of all strainshybridized with at least 9 of the 47 oligonucleotides. Depending on thecombination of spacer oligonucleotides hybridizing with thePCR-amplified DNA, 39 different "DVR types" of M. tuberculosis weredistinguished. This experiment shows that any M. tuberculosis strain canbe typed by this method, without the need to separate amplified M.tuberculosis DNA by electrophoresis. The 74 strains were also typed bythe classical IS6110 fingerprinting method as described (32) and 66different IS6110 types were distinguished. This indicates that the levelof strain differentiation using IS6110 fingerprinting is slightly highercompared to the method described in this invention. This method will bereferred to as "DVR-oligotyping". The method of DVR-oligotyping ishowever sufficiently specific to discern a large number of strainswithin a group such as M. bovis BCG and M. tuberculosis H37Rv and H37Ra.

Specificity of the DVR oligotyping method.

To determine whether the method of amplification and hybridisation isspecific for bacteria belonging to the M. tuberculosis complex, wesubjected 40 DNA samples originating from a wide variety ofmycobacterial species and other bacterial genera to the DVR oligotypingmethod. The target DNA's included the following bacterial species: E.coli, Bordetella pertassis, Afipia felis, Rochalimea lenselae,Mycobacterium avium. None of these targets led to a detectable positivehybridization reaction with any of the spacer oligonucleotides, herebyillustrating the specificity of the subject method.

Detection of M. tuberculosis in clinical specimens by DVR-oligotyping.

The sensitivity to detect M. tuberculosis by the above described methodwas tested by carrying out DVR-oligotyping with various amounts ofchromosomal DNA of strains H37Rv. M. bovis BCG and 2 clinical isolatesof M. tuberculosis. The sensitivity to detect DNA from each of thesestrains was at least 64 femtogram (fg) of DNA. 1 fg of chromosomal DNAcorresponds approximately to the quantity present in a single bacteriumand it is assumed, the DVR-oligotyping method will allow thesimultaneous detection and typing of DNA derived from a singlebacterium.

Furthermore, clinical sputa samples obtained from Dr. A. Kolk (RoyalTropical Institute, Amsterdam) were subjected to DVR-oligotyping, i.e.to the detection and differentiation methods according to the invention.All culture-positive samples were positive by DVR-oligotyping and theDVR type corresponded to the type as determined from purified DNAextracted from M. tuberculosis cultured from the corresponding sputumsamples.

MATERIALS AND METHODS

Determination of the DNA sequence of the DR region in H37Rv.

Cosmid T211, which contains a 35 Kb insert carrying the complete DRregion of strain H37Rv, was obtained from Dr S. Cole (Institut Pasteur,Paris). A physical map of cosmid T211 is shown in FIG. 6. MluI fragmentsof this cosmid were subcloned into MluI-cleaved DNA of plasmid pUCBM21.resulting in plasmids pPG11, pPG17 and pPG33 (FIG. 6). The latter 3plasmids were used to sequence the complete DR containing region ofstrain H37Rv. Sequencing was performed according to the dideoxy chaintermination method of Sanger et al. (28), using a 373A DNA Sequencer(Applied Biosystems, Fosre City, Calif. USA) following the protocolsprovided by the manufacturer).

Extraction of DNA from mycobacterial cells.

DNA was purified as described previously (33).

Bacterial strains used in this study.

Escherichia coli K12 strain DH5α (BRL, Maryland, USA) was used as a hostfor propagating plasmid pUCBM21 and derivatives. M. bovis BCG strain P3and M. tuberculosis H37Rv have been described previously (12, 15). Allother bacterial strains were clinical isolates, which were sent to theRIVM.

DVR oligotyping.

In vitro amplification of DNA.

10 Nanogram of purified M. tuberculosis DNA was added to a mixturecontaining 0.5 unit Super Tth polymerase (HT Biotechnology, Cambridge,UK). 5 μl of 10× concentrated Super Rth buffer (HT biotechnology,Cambridge, UK), 20 nMol of each dNTP, and 20 pMol of each of the primersDRa and DRb. The final volume was adjusted to 50 μl. this mixture wassubjected to 30 cycles of amplification using the following scheme: 1min. 96° C. 1 min. 55° C. and 30 sec. 72° C.

Reverse line blot hybridization.

Oligonucleotides with a 5' terminal amino group were linked covalentlyto activated Biodyne C membrane (18, 38). The Biodyne C membrane (PallBiosupport, Glen Cove, N.Y., USA) was activated by incubation for 10min. in 10 ml freshly prepared 16% (w/v) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The blot was rinsed withwater and placed immediately in a miniblotter system (Immunetics, ITKDiagnostics, Uithoorn, The Netherlands). Each slot of the miniblotterwas filled with 150 μl of a 0.125 μM oligonucleotide solution in 500 mMNaHCO₃, pH 8.4. After 1 min. incubation at room temperature, theoligonucleotide solutions were removed by aspiration. The filter wasremoved from the miniblotter, treated with 100 mM NaOH for 10 min. toinactivate the membrane and washed in 2×SSPE (360 mM NaCl, 20 mM NaH₂PO₄, 2 mM EDTA, pH 7.2). supplemented with SDS (0.1%) for 5 min. at 54°C. The filter was mounted into the miniblotter, in such a way that theslots were perpendicular to the line pattern of the appliedoligonucleotides. The slots of the miniblotter were filled with 150 μlof diluted, heat-denatured, biotin-labeled PCR products (20 μl PCRproduct diluted in 130 μl 2×SSPE, 0.1% SDS) and hybridized for 45 min at54° C. after emptying the slots by aspiration, the filter was washed in150 ml of 2×SSPE, 0.5% SDS for 10 min. at 54° C. and incubated in 10 mlof streptavidin-peroxidase conjugate (Boehringer, Mannheim) diluted1:4000 in 2×SSPE. (0.5% SDS) for 30 min. at 42° C. The filter was washedin 150 ml of 2×SSPE, 0.5% SDS for 10 min. at 42° C. and rinsed brieflyat room temperature with 150 ml of 2×SSPE. For chemiluminescentdetection of hybridizing DNA the filter was incubated in 10 ml ECLdetection liquid (Amersham, 's Hertogenbosch, The Netherlands) andexposed for one min. to X-ray film (Hyperfilm, Amersham).

BRIEF DESCRIPTION OF THE DRAWINGS

Legend to FIG. 1.

Structure of the DR region of M. bovis BCG and M. tuberculosis H37Rv.The rectangles depict the 36 bp DR sequences, which are interspersed byunique spacers varying 29 to 41 bp in size. The site of insertion of theIS6110 element in the DR region is depicted. Part of the DR region of M.bovis BCG has been sequenced previously (15) and this part is depictedin black. The non-sequenced part is in gray. The whole DR region ofH37Rv was sequenced as part of the invention. Gaps in the H37Rv sequenceindicate the absence of DVR's, which are present in M. bovis BCG.

Legend to FIG. 2.

Principle of the in vitro amplification of DNA within the DR region ofM. tuberculosis complex bacteria. The repeating units within the DRcluster are the DVR's. Each DVR is composed of a constant 36 base pairsequence, DR, and a variable part, the spacer (A, B, C and D,respectively). Four sequential DVR's are represented as DVR-A, DVR-B,DVR-C, and DVR-D. The use of the 2 primers, DRa and DRb (arrows a andb), having sequences based on the DR sequence, for in vitroamplification of DNA, will lead to the amplification of any DVR or astretch composed of a discrete number of neighbouring DVR's.

Legend to FIG. 3.

Nucleotide sequence of the DR region in M. tuberculosis strain H37Rv andthe regions flanking the DR region. Sequences homologous to the DRsequence are underlined, sequences used as oligonucleotides in the assayare printed in bold.

Legend to FIG. 4.

Gel electrophoresis of in vitro amplified M. tuberculosis DNA amplifiedby PCR using the primers DRa and DRb. Each lane was loaded withone-fifth of the total amount of amplified DNA from different clinicalM. tuberculosis isolates. The quantity of DNA used as a target for thePCR was 10 nanogram.

Legend to FIG. 5.

Hybridisation patterns of the in vitro amplified DVR products of 72different M. tuberculosis isolates and 5 different M. bovis BCG isolateswith 41 different oligonucleotides. The oligonucleotides used arederived from the spacer sequences 1 to 41 as described in Materials andMethods. The primers Dra and Drb (see FIG. 2) were used as drivers forthe in vitro amplification of the DVR's with the DR region.

The spacer oligonucleotides were covalently bound to a Biodyne C filterin a pattern of parallel lines and the hybridization with in vitroamplified DVR DNA was done in parallel channels perpendicular to thespacer oligonucleotide pattern as described in the materials andmethods. Strain 1: H37Rv; strain 41: H37Ra; strains 44-46: different M.bovis BCG isolates; strain 77: M. bovis BCG P3; all other strains:clinical isolates of M. tuberculosis.

Legend to FIG. 6.

Restriction map of the insert of cosmid T211, containing the complete DRregion of H37Rv; localisation of the MluI fragments subcloned into theplasmids pPG17, pPG11, and pPG33.

REFERENCES

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6. Daley, C. L. P. M. Small. G. F. Schecter, G. K. Schoolnik. R. A.McAdam, W. R. Jacobs, Jr., and P. C. Hopewell. 1992. An outbreak oftuberculosis with accelerated progression among persons infected withthe human immunodeficiency virus: an analysis using restriction fragmentlength polymorphisms. N. Engl. J. Med. 326:231-235.

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10. Fischl, M. A, R. B. Uttamchandani, G. L. Daikos, et al. 1992. Anoutbreak of tuberculosis caused by multiple-drug resistant tuberclebacilli among patients with HIV infection. Ann. Intern. Med.117:177-183.

11. Fomukong, N. G., J. W. Dale, T. W. Osborn and J. M. Grange. 1992.Use of gene probes on the insertion sequence IS986 to differentiatebetween BCG vaccine strains. J. Appl. Bacteriol. 72:125-133.

12. Groenen, P. M. A., A. E. van Bunschoten, D. van Soolingen, and J. D.A. van Embden. 1993. Nature of DNA polymorphism in the direct repeatcluster of Mycobacterium tuberculosis; Application for straindifferentiation by a novel method. Mol. Microbiol. (1993) 10 (5)1057-1065.

13. Haas, W. H., W. R. Butler, Ch. L. Woodley and J. T. Crawford. 1993.Mixed-linker polymerase chain reaction: a new method for rapidfingerprinting of isolates of the Mycobacterium tuberculosis Complex. J.Clin. Microbiol. 31:1293-1298.

14. Hermans, P. W. M., D. van Soolingen. J. W. Dale, A. R. Schuitema, R.A. McAdam, D. Catty, and J. D. A. van Embden. 1990. Insertion elementIS986 from Mycobacterium tuberculosis: a useful tool for diagnosis andepidemiology of tuberculosis. J. Clin. Microbiol. 28:2051-2058.

15. Hermans, P. W. M., D. van Soolingen. E. M. Bik, P. E. W. de Haas, J.W. Dale, and J. D. A. van Embden. 1991. The insertion element IS987 fromM. bovis BCG is located in a hot spot integration region for insertionelements in M. tuberculosis complex strains. Infect. Immun.59:2695-2705.

16. Hermans, P. W. M., D. van Soolingen, and J. D. A. van Embden. 1992.Characterization of a major polymorphic tandem repeat in Mycobacteriumtuberculosis and its potential use in the epidemiology of Mycobacteriumkansasii and Micobacterium gordonae. J. Bacteriol. 174:4157-4165.

17. Jeffreys, A. J., A. Macleod, K. Tamaki, D. L. Neil and D. G.Monckton. 1991. Minisatellite repeat coding as a digital approach to DNAtyping. 354: 204-209.

18. Kaufholt, A., A. Podbielski, G. Baumgarten, M. Blokspoel, J. Top,and L. Schouls. 1994. Rapid typing of group A streptococci by the use ofDNA amplification and non-radioactive allele specific oligonucleotideprobes. FEMS Microbiology Letters, in press.

19. Mazurek, G. H., M. D. Cave, K. D. Eisenach, R. J. Wallace JR, J. H.Bates, and J. T. Crawford. 1991. Chromosomal DNA fingerprint patternsproduced with IS6110 as strain specific markers for epidemiologic studyof tuberculosis. J. Clin. Microbiol. 29:2030-2033.

20. McAdam, R. A., P. W. M. Hermans, D. van Soolingen, Z. F. Zainuddin,D. Catty. J. D. A. van Embden, and J. W. Dale. 1990. Characterization ofa Mycobacterium tuberculosis insertion sequence belonging to the IS3family. Mol. Microbiol. 4:1607-1613.

21. Mendiola, M. V., C.. Martin, I. Otal, and B. Gicquel. 1992. Analysisof regions responsible for IS6110 RFLP in a single Mycobacteriumtuberculosis strain. Res. Microbiol. 143:767-772.

22. Otal, I., C. Martin, V. Vincent-Levy-Frebault. D. Thierry, and B.Gicquel. 1991. Restriction fragment length polymorphism analysis usingIS6110 as an epidemiological marker in tuberculosis. J. Clin. Microbiol.29:1252-1254.

23. Palittapongarnpim. P., S. Chomic, A. Fanning, and D. Kunimoto. 1993.DNA fingerprinting of Mycobacterium tuberculosis by ligation-mediatedpolymerase chain reaction. Nucl. Ac. Res. 3:761-762.

24. Palittapongarnpim. P., S. Chomic, A. Fanning. and D. Kunimoto. 1993.DNA fragment length polymorphism analysis of M. tuberculosis isolatingby arbitrarily primed polymerase chain reaction. J. Inf. Diseases167:975-978.

25. Palittapongarnpim, P. S., S. Rienthong, and W. Panbangred. 1993.Comparison of restriction fragment length polymorphism of M.tuberculosis isolated from cerebrospinal fluid and sputum: a preliminaryreport. Tubercle and lung disease 1993. 74, 204-207.

26. Ross, C., K. Raios. K. Jackson, and B. Dwyer. 1992. Molecularcloning of a hightly repeated element from Mycobacterium tuberculosisand its use as an epidemiological tool. J. Clin. Microbiol. 30:942-946.

27. Ross, B. C., and B. Dwyer. 1993. Rapid, simple method for typingisolates of Mycobacterium tuberculosis by using the polymerase chainreaction. J. Clin. Microbiol. 31:329-334.

28. Sanger, F., Nicklen, S. and Coulson, A. R. (1977) DNA Sequencingwith chain-termination inhibitors. Proc. Natl. Acad. Sci. U.S.A.74:5463-5467.

29. Small. P. M., R. W. Schafer. P. C. Hopewell, S. P. Singh, M. J.Murphy, E. Desmond. M. F. Sierra, and G. K. Schoolnik. 1993. Exogenousreinfection with multidrug-resistant M. tuberculosis in patients withadvanced HIV infection. N. Eng. J. Med. 328:1137-1144.

30. Thierry, D., M. D. Cave, K. D. Eisenach, J. T. Crawford, J. H.Bates. B. Gecquel. and J. L. Guesdon. 1990. IS610. an IS-like element ofM. tuberculosis complex. Nucleic Acids Res. 18:188.

31. Van Embden, J. D. A., D. van Soolingen, P. M. Small, and P. W. M.Hermans. 1992. Genetic markers for the epidemiology of tuberculosis.Res. Microbiol. 143: 385-391.

32. Van Embden, J. D. A., M. D. Cave, J. T. Crawford, J. W. Dale, K. D.Eisenach, B. Gicquel, P. W. M. Hermans, C. Martin, R. McAdam, T. M.Shinnick, and P. M. Small. 1993. Strain identification of Mycobacteriumtuberculosis by DNA fingerprinting; Recommendations for a standardizedMethodology J. Clin. Microbiol. 31:406-409.

33. Van Soolingen, D., P. W. M. Hermans, P. E. W. de Haas, D. R. Soll,and J. D. A. van Embden. 1991. The occurrence and stability of insertionsequences in Mycobacterium tuberculosis complex strains; evaluation ofIS-dependent DNA polymorphism as a tool in the epidemiology oftuberculosis. J. Clin. Microbiol. 29:2578-2586.

34. Van Soolingen, D., P. W. M. Hermans, P. E. W. de Haas, and J. D. A.van Embden. 1992. Insertion element IS1081-associated RestrictionFragment Length Polymorphism in Mycobacterium tuberculosis Complexspacies: a reliable tool for recognizing Mycobacterium bovis BCG. J.Clin. Microbiol. 30:1772-1777.

35. Van Soolingen, D., P. E. W. de Haas, P. W. M. Hermans, P. M. A.Groenen and J. D. A. van Embden. 1993. Comparison of various repetitiveDNA elements as genetic markers for strain differentiation andepidemiology of Mycobacterium tuberculosis. J. Clin. Microbiol.31:1987-1995.

36. Yuen L. K., B. C. Ross, K. M. Jackson, and B. Dwyer. 1992.Characterization of Mycobacterium tuberculosis strains from Vietnamesepatients by southern blot hybridization. J. Clin. Micro. 31: 1615-1618.

37. WHO. 1993. Tuberculosis in the present time: A global overview ofthe tuberculosis situation. Tuberculosis Unit, Division of CommunicableDiseases. Geneva.

38. Y. Zhang, M. Y. Coyne, S. G. Will, C. H. Levenson, and E. Kawasaki.1991. Single-base mutational analysis of cancer and genetic diseasesusing membrane bound modified oligonucleotides. Nucl. Acids Res.19:3929-3933. Oligonucleotides used for DVR oligotyping referring to inthe Sequence Listing

    __________________________________________________________________________           Sequence id.                                                             Spacer No.:            No.:                                                 __________________________________________________________________________    01     3         5' TTG TAC TGC AAC CCG GAA TTC TTG A 3'                         - 02          4     5' ATA GAG GGT CGC CGG TTC TGG ATC A 3'                   - 03          5     5' CCT CAT AAT TGG GCG ACA GCT TTT G 3'                   - 04          6     5' CCG TGC TTC CAG TGA TCG CCT TCT A 3'                   - 05          7     5' ACG TCA TAC GCC GAC CAA TCA TCA G 3'                   - 06          8     5' TTT TCT GAC CAC TTG TGC GGG ATT A 3'                   - 07          9     5' CGT CGT CAT TTC CGG CTT CAA TTT C 3'                   - 08          10    5' GAG GAG AGC GAG TAC TCG GGG CTG C 3'                   - 09          11    5' CGT GAA ACC GCC CCC AGC CTC GCC G 3'                   - 10          12    5' ACT CGG AAT CCC ATG TGC TGA CAG C 3'                   - 11          13    5' TCG ACA CCC GCT CTA GTT GAC TTC C 3'                   - 12          14    5' GTG AGC AAC GGC GGC GGC AAC CTG G 3'                   - 13          15    5' ATA TCT GCT GCC CGC CCG GGG AGA T 3'                   - 14          16    5' GAC CAT CAT TGC CAT TCC CTC TCC C 3'                   - 15          17    5' GGT GTG ATG CGG ATG GTC GGC TCG G 3'                   - 16          18    5' CTT GAA TAA CGC GCA GTG AAT TTC G 3'                   - 17        19    5' CGA GTT CCC GTC AGC GTC GTA AAT C 3'                     - 18        20    5' GCG CCG GCC CGC GCG GAT GAC TCC G 3'                     - 19        21    5' CAT GGA CCC GGG CGA GCT GCA GAT G 3'                     - 20        22    5' TAA CTG GCT TGG CGC TGA TCC TGG T 3'                     - 21        23    5' ACC GCA GAC GGC ACG ATT GAG ACA A 3'                     - 22        24    5' AGC ATC GCT GAT GCG GTC CAG CTC G 3'                     - 23        25    5' CCG CCT GCT GGG TGA GAC GTG CTC G 3'                     - 24        26    5' GAT CAG CGA CCA CCG CAC CCT GTC A 3'                     - 25        27    5' CTT CAG CAC CAC CAT CAT CCG GCG C 3'                     - 26        28    5' GGA TTC GTG ATC TCT TCC CGC GGA T 3'                     - 27       29        5' TGC CCC GGC GTT TAG CGA TCA CAA C 3'                  - 28       30        5' AAA TAC AGG CTC CAC GAC ACG ACC A 3'                  - 29       31        5' GGT TGC CCC GCG CCC TTT TCC AGC C 3'                  - 30       32        5' TCA GAC AGG TTC GCG TCG ATC AAG T 3'                  - 31       33        5' GAC CAA ATA GGT ATC GGC GTG TTC A 3'                  - 32       34        5' CGC GAA CTC GTC CAC AGT CCC CCT T 3'                  - 33       35        5' CGT GGA TGG CGG ATG CGT TGT GCC C 3'                  - 34      36         5' GAC GAT GGC CAG TAA ATC GGC GTG G 3'                  - 35      37         5' CGC CAT CTG TGC CTC ATA CAG GTC C 3'                  - 36      38         5' GGA GCT TTC CGG CTT CTA TCA GGT A 3'                  - 37      39         5' ATG GTG GGA CAT GGA CGA GCG CGA C 3'                  - 38      40         5' CGC AGA ATC GCA CCG GGT GCG GGA G 3'                  - 39      41         5' ATA TCG CCC GCC ACA CCA CAG CCA C 3'                  - 40      42         5' CGC CGA TGA CAG CTA TGT CCG AGT G 3'                  - 41      43         5' TTC GCG CGG TGT TTC GGC CGT GCC C 3'                  - B1      44         5' TTG ACC TCG CCA GGA GAG AAG ATC A 3'                  - B2      45         5' TCG ATG TCG ATG TCC CAA TCG TCG A 3'                  - B3      46         5' GAC ATG ACG GCG GTG CCG CAC TTG A 3'                  - B4      47         5' AAG TCA CCT CGC CCA CAC CGT CGA A 3'                  - B5      48         5' TCC GTA CGC TCG AAA CGC TTC CAA C 3'                  - B6      49         5' CGA AAT CCA GCA CCA CAT CCG CAG C 3'                  - DRa     50         5' CCG AGA GGG GAC GGA AAC 3'                            - DRb     51         5' GGT TTT GGG TCT GAC GAC 3'                         __________________________________________________________________________

All oligonucleotide sequences are derived from sequences or the DRregion in strain M. tuberculosis H37Rv, except for oligonucleotideswhich are printed in bold. Latter ones are derived from the M. bovis BCGsequence (15). The 5' termini or the spacer oligonucleotides were linkedto an amino-group in order to enable a covalent binding to the Biodyne CMembranes. All oligonucleotides were obtained from Applied BiosystemsIncorporated, Perkin Elmer B. V., Gouda, The Netherlands.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 51                                          - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5519 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: M tubercu - #losis strain H37Rv DR cluster                          flanking - #IS6110                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - ACGCGTATAT CGGTTTCCTA CACCAGGATT CACGAGGGCA CGCAACGTTG GC -             #GAGCGACC     60                                                                 - - TCATGGAGGT ATGGCGGGCG CCGATCATCG ATGACACCGT ACTTCGATTG AT -            #CGCGGACG    120                                                                 - - GTGTGGTCGA CACCCGGGCT TTCAGCAAGA ACTCCGACAC GGGGGCCGTC TT -            #CGCGACAC    180                                                                 - - GGGAAGCCAC ACGATCCATC GCGCGCGCCT TTTGTAATCG AATCGCACGA AC -            #CGCCACCT    240                                                                 - - ACATCAAAGG CGATCCTTAC CGATACACTT TTCAGTACGC CCTCGACTTG CA -            #ACTGCAAA    300                                                                 - - GCTCGTGCGT GTTATTCGAA GCCGGGGAAC CCGTCGNGGT CGTCGATATC AC -            #CTCCGAGC    360                                                                 - - CATCCGGAGC CTAAATGCCC ACTCGCAGCC GTGAGGAGTA CTTCAATCTC CC -            #GCTCAAAG    420                                                                 - - TGGACGAGTC CAGCGGCACT ATAGGCAAGA TGTTCGTCCT CGTAATATAC GA -            #CATCAGCG    480                                                                 - - ACAACCGGCG GCGGGCTTCA CTTGCGAAGA TCCTGGCCGG GTTTGGCTAT CG -            #CGTCCAAG    540                                                                 - - AGTCCGCATT CGAAGCGATG CTGACGAAGG GCCAGCTCGC GAAACTAGTT GC -            #ACGTATCG    600                                                                 - - ACCGCTTCGC CATCGACTGC GACAACATCC GGATCTATAA GATAAGAGGT GT -            #TGCGGCAG    660                                                                 - - TTACGTTCTA CGGAAGGGGA CGGCTTGTCA GCGCAGAGGA GTTTGTGTTC TT -            #TTGACATC    720                                                                 - - ATCAGCAGGC ATTGTTACCA CACGCTGGAC GAATTGTCCA TAGAGTCGTC AG -            #ACCCAAAA    780                                                                 - - CCCCGAGAGG GGACGGAAAC TTAAAACCGT GTTGTACTGC AACCCGGAAT TC -            #TTGAACGT    840                                                                 - - CGTCAGACCC AAAACCCCGA GAGGGGACGG AAACCATAGA GGGTCGCCGG TT -            #CTGGATCA    900                                                                 - - CGCTCCCCTA GTCGTGTCGT CAGACCCAAA ACCCCGAGAG GGGACGGAAA CT -            #TTTTGCCT    960                                                                 - - CATAATTGGG CGACAGCTTT TGACCAAGTC GTCAGACCCA AAACCCCGAG AG -            #GGGACGGA   1020                                                                 - - AACTCGCAAG CGCCGTGCTT CCAGTGATCG CCTTCTAGTC GTCAGACCCA AA -            #ACCCCGAG   1080                                                                 - - AGGGGACGGA AACAACACCT CAGTAGCACG TCATACGCCG ACCAATCATC AG -            #GTCGTCAG   1140                                                                 - - ACCCAAAACC CCGAGAGGGG ACGGAAACTT TTCTGACCAC TTGTGCGGGA TT -            #AGCGGGCT   1200                                                                 - - TAGGTCGTCA GACCCAAAAC CCCGAGAGGG GACGGAAACA CCAATGCGTC GT -            #CATTTCCG   1260                                                                 - - GCTTCAATTT CAGCCTGTCG TCAGACCCAA AACCCCGAGA GGGGACGGAA AC -            #CTGAGGAG   1320                                                                 - - AGCGAGTACT CGGGGCTGCC GTCTGCGCTG GTCGTCAGAC CCAAAACCCC GA -            #GAGGGGAC   1380                                                                 - - GGAAACGCGT GAAACCGCCC CCAGCCTCGC CGGGGCCGCC TAGGTCGTCA GA -            #CCCAAAAC   1440                                                                 - - CCCGAGAGGG GACGGAAACA CTCGGAATCC CATGTGCTGA CAGCGGATTC GC -            #ATGTCGTC   1500                                                                 - - AGACCCAAAA CCCCGAGAGG GGACGGAAAC CGGGCAGCGT TCGACACCCG CT -            #CTAGTTGA   1560                                                                 - - CTTCCGGGTC GTCAGACCCA AAACCCCGAG AGGGGACGGA AACCAGGTGA GC -            #AACGGCGG   1620                                                                 - - CGGCAACCTG GCGGCCACGG GTCGGTCGTC AGACCCAAAA CCCCGAGAGG GG -            #ACGGAAAC   1680                                                                 - - ATGGGATATC TGCTGCCCGC CCGGGGAGAT GCTGTCCGAG GTCGTCAGAC CC -            #AAAACCCC   1740                                                                 - - GAGAGGGGAC GGAAACTTCG TCGACCATCA TTGCCATTCC CTCTCCCCAC GT -            #GTCGTCAG   1800                                                                 - - ACCCAAAACC CCGAGAGGGG ACGGAAACTT GCGCCAACCC TTTCGGTGTG AT -            #GCGGATGG   1860                                                                 - - TCGGCTCGGG TCGTCAGACC CAAAACCCCG AGAGGGGACG GAAACCTTGA AT -            #AACGCGCA   1920                                                                 - - GTGAATTTCG CGGATCAGAC CCAAAACCCC GAGAGGGGAC GGAAACATTC GC -            #ACGAGTTC   1980                                                                 - - CCGTCAGCGT CGTAAATCGC CAGTCGTCAG ACCCAAAACC CCGAGAGGGG AC -            #GGAAACCC   2040                                                                 - - GGCAACAATC GCGCCGGCCC GCGCGGATGA CTCCGGTCGT CAGACCCAAA AC -            #CCCGAGAG   2100                                                                 - - GGGACGGAAA CCGCATGGAC CCGGGCGAGC TGCAGATGGT CCGGGAGGTC GT -            #CAGACCCA   2160                                                                 - - AAACCCCGAG AGGGGACGGA AACTGGATTG CGCTAACTGG CTTGGCGCTG AT -            #CCTGGTGG   2220                                                                 - - TCGTCAGACC CAAAACCCCG AGAGGGGACG GAAACTTGGA GCGTGTCACC GC -            #AGACGGCA   2280                                                                 - - CGATTGAGAC AAGTCGTCAG ACCCAAAACC CCGAGAGGGG ACGGAAACCC TC -            #AGCTCAGC   2340                                                                 - - ATCGCTGATG CGGTCCAGCT CGTCCGTGTC GTCAGACCCA AAACCCCGAG AG -            #GGGACGGA   2400                                                                 - - AACCCAACCT CACCGCCTGC TGGGTGAGAC GTGCTCGCCG CGAGTCGTCA GA -            #CCCAAAAC   2460                                                                 - - CCTGAACCGC CCCGGCATGT CCGGAGACTC CAGTTCTTGG AAAGGATGGG GT -            #CATGTCAG   2520                                                                 - - GTGGTTCATC GAGGAGGTAC CCGCCGGAGC TGCGTGAGCG GGCGGTGCGG AT -            #GGTCGCAG   2580                                                                 - - AGATCCGCGG TCAGCACGAT TCGGAGTGGG CAGCGATCAG TGAGGTCGCC CG -            #TCTACTTG   2640                                                                 - - GTGTTGGCTG CGCGGAGACG GTGCGTAAGT GGGTGCGCCA GGCGCAGGTC GA -            #TGCCGGCG   2700                                                                 - - CACGGCCCGG GACCACGACC GAAGAATCCG CTGAGCTGAA GCGCTTAGCG GC -            #GGGACAAC   2760                                                                 - - GCCGAATTGC GAAGGGCGAA CGCGATTTTA AAGACCGCGT CGGCTTTCTT CG -            #CGGCCGAG   2820                                                                 - - CTCGACCGGC CAGCACGCTA ATTAACGGTT CATCGCCGAT CATCAGGGCC AC -            #CGCGAGGG   2880                                                                 - - CCCCGATGGT TTGCGGTGGG GTGTCGAGTC GATCTGCACA CAGCTGACCG AG -            #CTGGGTGT   2940                                                                 - - GCCGATCGCC CCATCGACCT ACTACGACCA CATCAACCGG GAGCCCAGCC GC -            #CGCGAGCT   3000                                                                 - - GCGCGATGGC GAACTCAAGG AGCACATCAG CCGCGTCCAC GCCGCCAACT AC -            #GGTGTTTA   3060                                                                 - - CGGTGCCCGC AAAGTGTGGC TAACCCTGAA CCGTGAGGGC ATCGAGGTGG CC -            #AGATGCAC   3120                                                                 - - CGTCGAACGG CTGATGACCA AACTCGGCCT GTCCGGGACC ACCCGCGGCA AA -            #GCCCGCAG   3180                                                                 - - GACCACGATC GCTGATCCGG CCACAGCCCG TCCCGCCGAT CTCGTCCAGC GC -            #CGCTTCGG   3240                                                                 - - ACCACCAGCA CCTAACCGGC TGTGGGTAGC AGACCTCACC TATGTGTCGA CC -            #TGGGCAGG   3300                                                                 - - GTTCGCCTAC GTGGCCTTTG TCACCGACGC CTACGCGCAG GATCCTGGGC TG -            #GCGGGTCG   3360                                                                 - - CTTCCACGAT GGCCACCTCC ATGGTCCTCG ACGCGATCGA GCAAGCCATC TG -            #GACCCGCC   3420                                                                 - - AACAAGAAGG CGTACTCGAC CTGAAAGACG TTATCCACCA TACGGATAGG GG -            #ATCTCAGT   3480                                                                 - - ACACATCGAT CCGGTTCAGC GAGCGGCTCG CCGAGGCAGG CATCCAACCG TC -            #GGTCGGAG   3540                                                                 - - CGGTCGGAAG CTCCTATGAC AATGCACTAG CCGAGACGAT CAACGGCCTA TA -            #CAAGACCG   3600                                                                 - - AGCTGATCAA ACCCGGCAAG CCCTGGCGGT CCATCGAGGA TGTCGAGTTG GC -            #CACCGCGC   3660                                                                 - - GCTGGGTCGA CTGGTTCAAC CATCGCCGCC TCTACCAGTA CTGCGGCGAC GT -            #CCCGCCGG   3720                                                                 - - TCGAACTCGA GGCTGCCTAC TACGCTCAAC GCCAGAGACC AGCCGCCGGC TG -            #AGGTCTCA   3780                                                                 - - GATCAGAGAG TCTCCGGACT CACCGGGGCG GTTCACCCCG AGAGGGGACG GA -            #AACTCGGG   3840                                                                 - - GAGCCGATCA GCGACCACCG CACCCTGTCA GTCGTNAGAC CCAAAACCCC GA -            #GAGGGGAC   3900                                                                 - - GGAAACCTTC AGCACCACCA TCATCCGGCG CCTCAGCTCA GCATGTCGTC AG -            #ACCCAAAA   3960                                                                 - - CCCCGAGAGG GGACGGAAAC CCTTCGACGC CGGATTCGTG ATCTCTTCCC GC -            #GGATAGGT   4020                                                                 - - CGTCAGACCC AAAACCCCGA GAGGGGACGG AAACTGCCCC GGCGTTTAGC GA -            #TCACAACA   4080                                                                 - - CCAACTAATG GTCGTCAGAC CCAAAACCCC GAGAGGGGAC GGAAACCAGC GA -            #AATACAGG   4140                                                                 - - CTCCACGACA CGACCACAAC GCGTCGTCAG ACCCAAAACC CCGAGAGGGG AC -            #GGAAACTC   4200                                                                 - - TTGACGATGC GGTTGCCCCG CGCCCTTTTC CAGCCGTCGT CAGACCCAAA AC -            #CCCGAGAG   4260                                                                 - - GGGACGGAAA CAGGTTCGCG TCAGACAGGT TCGCGTCGAT CAAGTCCGGT CG -            #TCAGACCC   4320                                                                 - - AAAACCCCGA GAGGGGACGG AAACTTTATC ACTCCCGACC AAATAGGTAT CG -            #GCGTGTTC   4380                                                                 - - AAGTCGTCAG ACCCAAAACC CCGAGAGGGG ACGGAAACAT TTTGAGCGCG AA -            #CTCGTCCA   4440                                                                 - - CAGTCCCCCT TTCAGGTCGT CAGACCCAAA ACCCCGAGAG GGGACGGAAA CG -            #CCCCGTGG   4500                                                                 - - ATGGCGGATG CGTTGTGCGC GCAAGTGTCG TCAGACCCAA AACCCCGAGA GG -            #GGACGGAA   4560                                                                 - - ACCCGACGAT GGCCAGTAAA TCGGCGTGGG TAACCGATCC GGGTCGTCAG AC -            #CCAAAACC   4620                                                                 - - CCGAGAGGGG ACGGAAACTA GTACGCCATC TGTGCCTCAT ACAGGTCCAG TG -            #CCCTGTCG   4680                                                                 - - TCAGACCCAA AACCCCGAGA GGGGACGGAA ACCTGACGGC ACGGAGCTTT CC -            #GGCTTCTA   4740                                                                 - - TCAGGTAGTC GTCAGACCCA AAACCCCGAG AGGGGACGGA AACCCTCATG GT -            #GGGACATG   4800                                                                 - - GACGAGCGCG ACTATCGGGG TCGTCGGACC CAAAACCCCG AGAGGGGACG GA -            #AACTGGAC   4860                                                                 - - GCAGAATCGC ACCGGGTGCG GGAGGTGCAG CAGTCGTCAG ACCCAAAACC CC -            #GAGAGGGG   4920                                                                 - - ACGGAAACGC ATATCGCCCG CCACACCACA GCCACGCTAC TGCTCCATGT CC -            #TCAGACCC   4980                                                                 - - AAAACCCCGA GAGGGGACGG AAACACACCG CCGATGACAG CTATGTCCGA GT -            #GACATCCT   5040                                                                 - - CCCAGTCGTC AGACCCAAAA CCACGAGAGG GGACGGAAAC TTGAACCGCC CT -            #TCGCGCGG   5100                                                                 - - TGTTTCGGCC GTGCCCGAGT CGTCAGACCC AAAACCCCGA GAGGGGACGG AA -            #ACTACGAC   5160                                                                 - - GACTGGGTCG CCACCGCGTC TGTCGACCTN GCATTCAGGA TGANCATGAT GG -            #CGGCGTTG   5220                                                                 - - ACGGTGAGGA CGTTTGGTCA TGAAATGNNC NCGCCGGGAG ATGTCCGGCG GG -            #GTCGGTGG   5280                                                                 - - TGTTCGGGGT GTCGGTGTGG TGTTCAGTCT GCCGTGACTT CGGCGATGGC GG -            #TGCGGNTG   5340                                                                 - - GTGGATTCGT CGACGATGGC CTTNTCGGCG GCGAAGGCGG CGACGAGGGC TT -            #NCAGGGCG   5400                                                                 - - AGGTTGTTGA CCGCGCGGGG GTAGCCCCGN CTGGTCTGGT GGATCAACCC GA -            #TGGCGTCG   5460                                                                 - - TCGGAGAACA GGGCATCGTC GNGTCCGGCT ANCTTNAGGT TGTTGCGTAG GT -            #AGCTTCC    5519                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Consensus - #sequence of direct repeat of M.                        tuberculosis                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - GTCGTCAGAC CCAAAACCCC GAGAGGGGAC GGAAAC      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 01                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - TTGTACTGCA ACCCGGAATT CTTGA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 02                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - ATAGAGGGTC GCCGGTTCTG GATCA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 03                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - CCTCATAATT GGGCGACAGC TTTTG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 04                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - CCGTGCTTCC AGTGATCGCC TTCTA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 05                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - ACGTCATACG CCGACCAATC ATCAG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 06                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - TTTTCTGACC ACTTGTGCGG GATTA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 07                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - CGTCGTCATT TCCGGCTTCA ATTTC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 08                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - GAGGAGAGCG AGTACTCGGG GCTGC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 09                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - CGTGAAACCG CCCCCAGCCT CGCCG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 10                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - ACTCGGAATC CCATGTGCTG ACAGC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 11                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - TCGACACCCG CTCTAGTTGA CTTCC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 12                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                          - - GTGAGCAACG GCGGCGGCAA CCTGG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 15:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 13                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                          - - ATATCTGCTG CCCGCCCGGG GAGAT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 16:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 14                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                          - - GACCATCATT GCCATTCCCT CTCCC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 17:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 15                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                          - - GGTGTGATGC GGATGGTCGG CTCGG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 16                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                          - - CTTGAATAAC GCGCAGTGAA TTTCG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 19:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 17                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #19:                          - - CGAGTTCCCG TCAGCGTCGT AAATC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 20:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 18                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #20:                          - - GCGCCGGCCC GCGCGGATGA CTCCG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 21:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 19                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #21:                          - - CATGGACCCG GGCGAGCTGC AGATG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 22:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 20                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #22:                          - - TAACTGGCTT GGCGCTGATC CTGGT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 23:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 21                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                          - - ACCGCAGACG GCACGATTGA GACAA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 24:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 22                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #24:                          - - AGCATCGCTG ATGCGGTCCA GCTCG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 25:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 23                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                          - - CCGCCTGCTG GGTGAGACGT GCTCG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 26:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 24                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #26:                          - - GATCAGCGAC CACCGCACCC TGTCA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 27:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 25                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #27:                          - - CTTCAGCACC ACCATCATCC GGCGC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 28:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 26                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #28:                          - - GGATTCGTGA TCTCTTCCCG CGGAT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 29:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 27                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #29:                          - - TGCCCCGGCG TTTAGCGATC ACAAC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 30:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 28                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #30:                          - - AAATACAGGC TCCACGACAC GACCA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 31:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 29                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #31:                          - - GGTTGCCCCG CGCCCTTTTC CAGCC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 32:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 30                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #32:                          - - TCAGACAGGT TCGCGTCGAT CAAGT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 33:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 31                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #33:                          - - GACCAAATAG GTATCGGCGT GTTCA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 34:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 32                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #34:                          - - CGCGAACTCG TCCACAGTCC CCCTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 35:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 33                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #35:                          - - CGTGGATGGC GGATGCGTTG TGCGC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 36:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 34                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #36:                          - - GACGATGGCC AGTAAATCGG CGTGG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 37:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 35                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #37:                          - - CGCCATCTGT GCCTCATACA GGTCC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 38:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 36                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #38:                          - - GGAGCTTTCC GGCTTCTATC AGGTA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 39:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 37                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #39:                          - - ATGGTGGGAC ATGGACGAGC GCGAC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 40:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 38                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #40:                          - - CGCAGAATCG CACCGGGTGC GGGAG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 41:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 39                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #41:                          - - ATATCGCCCG CCACACCACA GCCAC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 42:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 40                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #42:                          - - CGCCGATGAC AGCTATGTCC GAGTG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 43:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer 41                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #43:                          - - TTCGCGCGGT GTTTCGGCCG TGCCC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 44:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    bovis BCG - #, spacer B1                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #44:                          - - TTGACCTCGC CAGGAGAGAA GATCA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 45:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    bovis BCG - #, spacer B2                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #45:                          - - TCGATGTCGA TGTCCCAATC GTCGA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 46:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    bovis BCG - #, spacer B3                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #46:                          - - GACATGACGG CGGTGCCGCA CTTGA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 47:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    bovis BCG - #, spacer B4                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #47:                          - - AAGTCACCTC GCCCACACCG TCGAA          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 48:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    bovis BCG - #, spacer B5                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #48:                          - - TCCGTACGCT CGAAACGCTT CCAAC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 49:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    bovis BCG - #, spacer B6                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #49:                          - - CGAAATCCAG CACCACATCC GCAGC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 50:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer DRa                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #50:                          - - CCGAGAGGGG ACGGAAAC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 51:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: primer fo - #r M.                                                    tuberculosis - #, spacer DRb                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #51:                          - - GGTTTTGGGT CTGACGAC             - #                  - #                      - #  18                                                                 __________________________________________________________________________

What is claimed is:
 1. In a method of in vitro amplification of nucleicacid using amplification primers in amplification reactions, theimprovement comprising using a pair of primers comprisingoligonucleotide sequences complementary to a part of a Direct Repeatsequence of a microorganism belonging to the M tuberculosis complex ofmicroorganisms, whereby hybridization to a Direct Repeat occurs andsubsequently elongation of the hybridized primer takes place, saidprimers being such that elongation in the amplification reaction occursfor one primer in a 5' direction and for the other primer in a 3'direction.
 2. A method according to claim 1, wherein said primers haveoligonucleotide sequences complementary to non-overlapping parts of theDirect Repeat sequence and such that the elongation reactions from eachprimer can occur without hindrance of the other when both primershybridize to the same Direct Repeat and undergo elongation.
 3. A methodaccording to claim 1, wherein one primer DRa is capable of elongation inthe 5' direction and another primer DRb is capable of elongation in the3' direction and DRa is complementary to a sequence of the Direct Repeatlocated to the 5' side of the sequence of the Direct Repeat to which DRbis complementary.
 4. A method according to claim 1, wherein themicroorganism is M tuberculosis, M bovis or M africanum.
 5. A methodaccording to claim 1, wherein the primer has an oligonucleotide sequencecapable of hybridizing to a consensus sequence of the Direct Repeatconsisting of SEQ ID NO:2 in such a manner that amplification occursunder the circumstances of the amplification reaction used.
 6. A methodaccording to claim 3, wherein the primer DRa consists of SEQ ID NO:50and DRb consists of SEQ ID NO:51.
 7. A method of detection of amicroorganism belonging to the M tuberculosis complex of microorganismscomprising1) amplifying nucleic acid from a sample by the methodaccording to claim 1, followed by 2) carrying out a hybridization test,wherein the amplification product is hybridized to an oligonucleotideprobe or a plurality of different oligonucleotide probes, eacholigonucleotide probe being homologous to a part of a spacer of theDirect Region of a microorganism belonging to the M tuberculosis complexwhereby hybridization occurs to the amplified product if a nucleic acidsequence of said spacer was present in the sample prior toamplification, and 3) detecting any hybridized products.
 8. A methodaccording to claim 7, wherein said hybridization step is carried outwithout prior electrophoresis or separation of the amplified product. 9.A method according to claim 7, wherein the hybridization test is carriedout using a number of oligonucleotide probes, said number comprising atleast a number of oligonucleotides probes specific for the totalspectrum of microorganisms it is desired to detect.
 10. A methodaccording to claim 7, wherein the microorganism belongs to the group Mtuberculosis or M bovis or M africanum.
 11. A method according to claim7, wherein the oligonucleotide probe is at least seven oligonucleotideslong and is a sequence complementary to a sequence selected from any ofspacers 1-43 of M tuberculosis H37Rv and spacers 1-49 of M bovis BCG oris a sequence complementary to fragments or derivatives of said spacers,said oligonucleotide probe being capable of hybridizing to one of saidspacers and comprising at least seven consecutive nucleotides homologousto one of said spacers.
 12. A method according to claim 7, wherein theoligonucleotide probe is at least 7 oligonucleotides long and is asequence complementary to any spacer common to M tuberculosis H37Rv andM bovis BCG, said oligonucleotide probe being capable of hybridizing tosaid spacer and comprising at least seven consecutive nucleotidesexhibiting at least 60% homology to said spacer.
 13. A method fordifferentiating the type of microorganisms belonging to the Mtuberculosis complex in a sample, comprising carrying out the methodaccording to claim 7, followed by comparison of the hybridizationpattern obtained with a reference.
 14. A method for differentiating thetype of microorganism belonging to the M tuberculosis complex in asample according to claim 13 wherein said reference is the hybridizationpattern obtained with at least one strain of microorganism belonging tothe M tuberculosis complex of microorganisms.
 15. A method fordifferentiating the type of microorganism belonging to the Mtuberculosis complex in a sample according to claim 13 wherein saidreference is a source providing a list of spacer sequences and sourcesthereof.
 16. A pair of primers wherein both primers compriseoligonucleotide sequences of at least 7 oligonucleotides and arecomplementary to a part of a Direct Repeat sequence of a microorganismbelonging to the M tuberculosis complex of microorganisms, wherebyhybridization occurs and subsequently elongation of the hybridizedprimer takes place, said primers being such that elongation in theamplification reaction occurs for one primer in the 5' direction and forthe other primer in the 3' direction and wherein said oligonucleotidesequence comprises at least seven consecutive nucleotides homologous tosaid Direct Repeat sequence.
 17. Primer pair according to claim 16,comprising one primer DRa capable of elongation in the 5' direction andanother primer DRb capable of elongation in the 3' direction with DRabeing complementary to a sequence of the Direct Repeat located to the 5'side of the sequence of the Direct Repeat to which DRb is complementary,the Direct Repeat being present in the Direct Region of a microorganismbelonging to the group of M tuberculosis complex.
 18. Primer pairaccording to claim 17, wherein DRa has sequence id. no. 50 and DRb hassequence id. no.
 51. 19. Kit for carrying out a method of in vitroamplification of nucleic acid or for differentiating the type ofmicroorganism belonging to the M tuberculosis complex in a sample,comprising a primer pair according to claim 16, and an oligonucleotideprobe or a carrier, said carrier comprising at least 1 oligonucleotideprobe specific for a spacer region of a microorganism of the groupbelonging to M tuberculosis complex, said oligonucleotide probe being anoligonucleotide probe of at least 7 nucleotides, said probe beinghomologous to any of the spacer sequences id no. 1-23 and 44-49 of Mbovis BCG and id no. 35-43 of M tuberculosis H37Rv or to fragments orderivatives of said spacer sequences thereby to hybridize to said spacersequence, said oligonucleotide probe comprising at least sevenconsecutive nucleotides homologous to said spacer sequence.
 20. Kit forcarrying out a method of in vitro amplification of nucleic acid or fordifferentiating the type of microorganism belonging to the Mtuberculosis complex in a sample, comprising a primer pair according toclaim 16, and a plurality of oligonucleotide probes or a carrier, saidcarrier comprising at least 1 oligonucleotide probe specific for aspacer region of a microorganism of the group belonging to Mtuberculosis complex, said oligonucleotide probe being anoligonucleotide probe of at least 7 nucleotides, said probe beinghomologous to any of the spacer sequences id no. 1-23 and 44-49 of Mbovis BCG and id no. 35-43 of M tuberculosis H37Rv or to fragments orderivatives of said spacer sequences thereby to hybridize to said spacersequence, said oligonucleotide probe comprising at least sevenconsecutive nucleotides homologous to said spacer sequence.